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Hoefer SE900
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1. Fig 12 Plate rack SE914 Separator Sheets The sheets have a protective film on both sides which should be removed before use Wonder Wedge The Wonder Wedge is helpful to pry apart the glass plates after electrophoresis Gel Seal Gel seal is used in the separation tank to lubricate the channels for the PAGE rack and help it slide into place If the PAGE rack starts to stick as it is inserted into the tank apply a thin layer of gel seal to the inside of the four channels with a gloved finger Gel seal is also used on the gasket in the multiple gel caster Plate Rack The plate rack SE914 may be ordered separately It is very conve nient to hold the glass cassettes during assembly of the multiple gel caster and when applying IPG strips to the top of the second dimen sion gels e p9 Note Do not remove the layer of gel seal from inside the white channels that position the PAGE rack Note Do not run the pumps dry Fig 13 Panel located on lower right side of the SE900 The power supply plugs into the power supply inlet on the right The ON OFF toggle switch is on the left e p10 Operating Instructions Separation Tank Setup 1 Position the SE900 with the black safety interlock on the left as shown in Fig 20 on page 17 Place the separation tank near a sink for easy buffer draining and disposal Before using for the first time disassemble the unit and wash with a dilute solution of a laborat
2. p12 Prepare the Acrylamide Solution See Appendix A for SDS PAGE recipes For casting six SE9102 1 1 0 gels 400 ml is needed De aerate the gel solution for 5 minutes Add the initiator 10 w v APS and the catalyst 10 v v TEMED just prior to casting the gels Pour the gel solution into the channel in the back of the caster The solution will flow down the channel and fill the casssettes from the bottom Fig 15 The TEMED is diluted to 10 for better distribution during mixing of the gel solutions Pour the solution as a slow continuous stream Try to minimize the introduction of air bubbles into the flow Fill solution to 0 5 1 cm below the top of the glass plate to allow room for the IPG strip and an agarose seal Overlay each gel with a thin layer of water saturated n butanol or diluted gel buffer to get the best surface on the top of the gel S owly apply the overlay near the gel surface from one side taking care to prevent mixing Allow the overlay to flow across the surface unaided Fig 16 Allow the gel to polymerize approximately one hour Remove the overlay by rinsing the top of the gel several times with distilled water Use diluted gel buffer as a final rinse Optional storage The second dimension gels can be temporarily stored in the caster The second dimension gels can also be removed from the caster and stored 1 2 days submerged in gel buffer before use There is a risk that with prolonged storage
3. re pas sp cifi par Hoefer Inc la protection fourni par l quipement pourrait tre diminu e Cet instrument est concu pour l usage de laboratoire int rieur seulement Seulement les accessoires et les parties ont approuv ou ont fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entretenir ce produit utilise Seulement une alimentation qui est CET a marqu ou la s curit certifi par un nationalement reconnu essayant le laboratoire Le couvercle de s curit doit tre sa place avant connecter l alimentation mene une alimentation Tourner tous contr les d alimentation de et d brancher les avances de pouvoir avant enlever le couvercle de s curit Circuler seulement de l eau ou 50 50 glycol d eau thyl ne par l exchanger de chaleur si si quip Ne pas connecter l exchanger de chaleur un robinet d eau ou la source d agent de refroidissement o la pression d eau est non r gul e Ne Jamais introduire d antigel ou du dissolvant organique dans n importe quelle partie de l instrument Les dissolvants organiques causeront des dommages irr parables l unit Ne pas fonctionner avec les temp ratures de tampon au dessus du maximum a sp cifi des sp cifications techniques La surchauffe causera des dommages irr parables l unit Wichtige Informationen German Wenn diese Ausr stung gewisserma en nicht angegeben durch Hoefer Inc verwendet wird kann der du
4. the gels abosrb water and the cassette partially opens leading to skewed dye fronts Note It is a good idea to practice with water and the peristaltic pump to empirically determine the volumes and the flow rates to cast good gradient gels Note Avoid getting air bubbles into the pump tubing Air bubbles will interfere with proper gradient formation Note The displacement solution is required The gradient will not form correctly without displacing the acrylamide up inside the cassettes It also helps keep acrylamide from polymerizing in the tubing and in the bottom of the caster simplifying clean up The displacement solution should not enter the bottom of the cassettes Gradient Gels Linear gradient gels can be cast using an optional accessory the Hoefer SG500 Gradient Maker The G500 mixes low percentage and high percentage gel solutions that are pumped into the port at the bottom of the multiple gel caster using a peristaltic pump Pouring a Linear Gradient Gel 1 Assemble the multiple gel caster as described on page 11 with the following two exceptions e Do not insert the triangular rubber gasket at the bottom of the gel caster Remove the cap from the bottom inlet port 2 Attach one end of laboratory grade tubing to the SG500 outlet port Insert the tubing through a peristaltic pump and attach the other end to the inlet port of the SE915 multiple gel caster faceplate 3 Calculate the volume of monomer so
5. 2 0 2 0 2 0 Deionized H20 112 2 95 5 79 0 62 4 45 8 29 1 10 APS Soln 4 2 0 2 0 2 0 2 0 2 0 2 0 10 TEMED Soln 5 0 50 0 46 0 34 0 27 0 23 0 20 Final Volume 200 0 200 0 200 0 200 0 200 0 200 0 200 ml HEAVY 7 5 10 0 12 5 15 0 17 5 20 Acrylamide Stock Soln 1 50 0 66 7 83 3 100 0 116 7 133 3 1 5 M TrisCl 50 0 50 0 50 0 50 0 50 0 50 0 pH 8 8 Soln 2 10 SDS Soln 3 2 0 2 0 2 0 2 0 2 0 2 0 Deionized H20 83 0 66 5 49 9 33 3 16 6 0 0 10 APS Soln 4 2 0 2 0 2 0 2 0 2 0 2 0 10 TEMED Soln 5 0 46 0 34 0 27 0 23 0 20 0 17 Glycerol 12 50 12 50 12 50 12 50 12 50 12 50 Final Volume 200 0 200 0 200 0 200 0 200 0 200 0 e p29 e p30 Appendix B References Denaturing Gel Systems Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T Nature 227 680 685 1970 Two dimensional Electrophoresis Anderson Leigh and Anderson Norman G High resolution two dimensional electrophoresis of human plasma proteins Proc Natl Acad Sci USA 74 5421 5425 1977 Anderson L Tiwo Dimensional Electrophoresis Operation of the ISO DALT System Second Edition Large Scale Biology Press 1991 O Farrell P H High resolution two dimensional electrophoresis of proteins J Biol Chem May 25 250 10 4007 4021 1975 Bjellqvist B et al Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6
6. 317 339 1982 Gorg A et al The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 1988 G rg A Two dimensional electrophoresis with immobilized pH gradients current state Biochem Soc Trans 21 130 132 1993 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 429 5732 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc ISO DALT is a trademark of Large Scale Biology Corporation 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
7. Appendix A for suggested recipe Avoid intro dimension gel ducing air bubbles while applying the overlay 4 Allow the agarose to solidify before placing gels into the separation tank Fig 18 Adding agarose overlay e p15 Caution Do not run the circulation pumps without buffer in the separation tank Caution Glass cassettes will not easily slide into a dry tank Fill with buffer first and use water or buffer to help lubricate the glass cassettes and rubber flaps Fig 19 Gel orientation e p16 Page Separation Remove the safety lid 2 Fill the tank with 12 liters of 1X electrophoresis buffer This can be prepared in the separation tank by adding 10X stock and water Gently raise and lower the PAGE rack to aid in mixing in buffer 3 Turn on the circulation pumps The toggle switch is located on the lower right side of the SE900 4 Fully insert the PAGE rack to the bottom of the separation tank making sure the keying features are aligned 5 Insert gels into the slots in the PAGE rack There is one specific orien tation for the gel cassettes The rubber hinge should be on the bottom of the separation tank The sealed IPG strip should be on the left the side with the black safety interlock and the black high voltage lead in the safety lid Fig 18 6 Check that both ends of the gel are exposed to the buffer The glass plates should be centered Fig 19 Good positioning centered between flaps Bad position
8. Oververhittend zal onherstelbare schade aan de eenheid veroorzaken T rke Tietoa Finnish Jos t t varusteita k ytet n tavassa ei m ritetty Hoefer Inc suojelu ehk isty varusteille saattaa olla avuton T m v line suunnitellaan sis laboratoriok yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoefer Inc oheen voi k ytt k ytt miselle valvoalle ja servicing t m tuote e Vain k ytt k ytt j nnitett joka on CE merkitsi tai turvallisuus joka on todistanut aidoksi ohi joka on kansallisesti tunnustettnut testaaminen laboratoriota Turvallisuuskansi t ytyy olla paikallaan ennen yhdist minen e pii kayttdjannitelyijyja k ytt j nnitteeseen Kiert kaikki k ytt j nnitevalvonnat ja irrottaa valtalyijyt ennen poistaminen turvallisuuskantta Kiert vain vesi tai 50 50 vesi ethylene glycol siin tapauksessa varustetun lamm nvaihtimen l pi l yhdist l mm nvaihdinta vesinapautukseen eik j hdytysnestel hteeseen miss vesipaine on unregulated Pakkasneste eik orgaaninen liuotin v lineen osassa ei esitele Koskaan Orgaaniset liuottimet aiheuttavat korvaamattoman vahingon yksikk n Ei k yt puskuria yll olevia l mp tiloja enint n m ritetyill teknisill t smennyksill Ylikuumeneminen aiheuttaa korvaamattoman vahingon yksikk n Information Importante French Si cet quipement est utilis dans une mani
9. i sa utrustad fall Inte kopplar varmen exchanger till en vatten kran eller nagot kylmedel k lla dar vattnet trycket ar unregulated Infor aldrig kylvatska eller n got organiska l sningsmedel in i nagon del av instrumentet Organiskt l sningsmedel ska orsaka irreparable skada till enheten Anv nd inte med buffert temperaturer ver det h gsta angivna tekniska specifikationerna Overhettning skulle orsaka irreparabla skador p enheten piv English French German Italian Spanish Swedish La Lo La La La Waste Electrical And Electronic Equipment WEEE This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obtenir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Hausm ll entsorgt werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten d
10. multiple gel caster is included with the SE900 1 0 Fig 10 and can be ordered separately as the SE915 The multiple gel caster is used to cast up to seven 1 mm thick gels at the same time Separator sheets are placed between glass cassettes before casting to keep the cassettes from sticking to each other after polymerization When casting single percentage gels by hand pour the gel solution down the channel in the back wall of the caster Fig 11 The cassettes will fill from the bottom producing better quality gels and reducing the chances of trapping air bubbles When casting gradient gels the gel solution should be slowly pumped in through the port at the bottom The triangular plug at the bottom should be removed The triangular region forms a space for the gradi ent gel to spread out and enter the glass cassettes uniformly When using butanol overlays try to minimize contact of butanol with the plastic multicaster Prolonged contact with butanol may craze the plastic of the multicaster Space Saver The space saver is used to fill space within the multiple gel caster and reduce the amount of gel solution One space saver is equivalent in thickness to one 1 0 mm glass cassette When casting six 1 0 mm thick gels use one space saver at the back of the multiple gel caster One space saver is included with the multiple gel caster Additional space savers SE912 can be ordered separately if less than 6 gels are routinely cast
11. tank with deionied water turn on pumps and circulate for 5 10 minutes Turn off pumps and drain again Remove the PAGE rack and allow the components to air dry Remove and rinse any residual acrylamide from the drain filter at the bottom of the separation tank Replace the drain filter so that it is below flush Care and Maintenance Handle the PAGE rack with care to prevent damage to the banana plugs and electrode wires Do not drop items onto the ceramic plate at the bottom of the separation tank f necessary clean the tank with mild detergent and rinse with distilled water Allow to air dry Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS 35 Rinse thoroughly with tap and distilled water Handle the plates with care to prevent chipping and do not pull on or stress the rubber hinge Do not autoclave any components of the system Do not heat any part above 45 C Do not use organic solvents abrasives strong cleaning solutions or strong acids or bases to clean the chambers If the PAGE rack starts to stick as it is inserted into the tank apply a thin layer of gel seal to the inside of the four channels with a gloved finger Technical Service and Repair Hoefer Inc offers complete technical support for all of our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Hoefe
12. the gels correctly within the gel tank For safety reasons the top of the safety interlock secures the lid during electrophoresis and the bottom prevents access to the drain port This prevents electrical hazard of draining electrified buffer A key at the bottom of the separation tank Fig 4 aligns with a mating feature in the PAGE rack Fig 6 to ensure proper orientation A drain port is included at the bottom behind the black safety inter lock The drain is protected by a removable filter that will block parts of gels from clogging the drain port This filter should be removed and rinsed off periodically e pd Fig 5 PAGE rack Fig 6 Orientation feature Fig 7 Banana plug p6 PAGE Rack The rack serves three purposes 1 It divides the tank into anodic and cathodic chambers and supports and seals the gel cassettes vertically inside the separation tank with rubber gaskets 2 It holds the platinum wire electrodes that conduct the current during electrophoresis 3 Features in the base create the circulation pattern to maintain constant temperature and provide efficient cooling if the SE900 is connected to a recirculating water bath The PAGE rack locates in four channels in the clear sides of the tank The PAGE rack has only one proper orientation cutout in the base Fig 6 aligns with a key in the separation tank If the PAGE rack is inserted incorrectly the buffer circulation will not function
13. Qus Hoefer SE900 Second Dimension Electrophoresis System OET A Hoefer Page Finder Important InformatlOR seet cerit narratur a rnt mne mne uncus ii Waste Electrical And Electronic Equipment WEEE vi FUNCTION and Description incaico 1 SPECICATION iii reet ta dpa m gut dendi 2 Unpacking and Inventon cn aane aeii 3 Operating Instt ctlons u eiii tete babeo Dai ads 10 Using the Multiple Gel Casier 11 IPG Strip Eeler arar ross 14 Page Separation eade tied fob ee Pies dub wd suede 16 Care and Maintenance sssi inise mite ce epist vnda 19 Technical Service and Repair 19 leefeg Tel 20 Ordering Information ccooccccocncconnncncnncnonnnnnononnnnnononnnoncncnncnnnnns 23 Appendix A Theory and Reclpes 24 SNL 25 Gel RECIPES suit nan d ele neci eei gedoe di 28 Appendix B References nennen nnn 30 Important Information English If this equipment is used in a manner not specified by Hoefer Inc the protection provided by the equipment may be impaired This instrument is designed for indoor laboratory use only Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Only use a power supply that is CE marked or safety certified by a nationally recognized testing laboratory The safety lid must be in place before connecting the power supply leads to a power supply Turn all power supply controls off and disconnect t
14. ak koli chladic kapaliny zdroje kde tlak vody je neregulo Nikdy zav st prost edek proti zamrznut nebo jak koli organick rozpou t dla do jak koli sti z tohoto n stroje Rozpustidl m zp sob nenapraviteln po kozen jednotka Nejsou provozov na s pufru teplot ch nad maxim ln stanovenou technick mi specifikacemi P eh t zp sob nenapraviteln po kozen jednotka Vigtig Information Danish Hvis dette udstyr bruges i en made ikke specificeret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan maske sv kkes Dette instrument er designet for indend rs laboratoriumbrug bare Bare tilbeh r og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funktionsfejl og betjening dette produkt bruger Bare en str mforsyning der er CE markerede eller sikkerhed som er blevet attesteret af en som nationalt er blevet anerkendt pr ve laboratorium Sikkerhedl get m v re p plads f r forbinding str mforsyningsblyet til en str mforsyning Drejer alle str mforsyningskontroller af og afbryder kraftblyet f r fjerning sikkerhedlaget Cirkulerer bare vand eller 50 50 vand ethylene glykol gennem varmeveksleren i s fald udrustet Forbind ikke varmeveksleren til en vandhane eller nogen k lemiddelkilde hvor vandtrykket er unregulated Introducerer Aldrig antifreeze eller noget organisk opl sningsmiddel ind i nogen del af instrumentet Organisk
15. ator sheet over the space saver Place a glass cassette on top of the first separator sheet O ON 00 A Place a separator sheet over the glass cassette 10 Continue to stack alternating glass cassettes and separator sheets for each gel being cast 11 Complete the assembly by adding enough separator sheets to fill the caster just above flush 12 Lightly grease the gasket in the faceplate with gel seal to assure a leak free casting Replace the gasket in the faceplate without bunching or stretching the gasket 13 Complete the assembly with a separator sheet 14 Slide the faceplate into place under the two screws and tighten the screws 15 Attach the 6 red clamps 3 per side along the sides of the caster 16 Make sure the cap is on the bottom port of the caster before pouring gels by hand 17 Stand the assembled caster upright on a level surface Face Plate Glass Cassette Separator Sheet Space Saver Red Clamp Multiple Gel Caster e p11 1 2 Caution Acrylamide is a neurotoxin Extreme care should be used when handling and preparing acrylamide solutions 3 4 5 Fig 15 Pour the solution in a slow continuous stream 8 Fig 16 Applying overlay Note A stacking gel can be used if desired but is not necessary Note It is necessary to remove any butanol overlays before storing the gels in the caster Leaving butanol in the caster can lead to whitening and brittleness of the acrylic
16. bbles between the strip and the second dimension gel p22 Ordering Information Product Quantity Order Code Large Format PAGE 1 SE900 Large Format PAGE Complete 1 SE900 1 0 Tubing Kit for Recirculating Chiller 1 SE908 Multicaster 1 SE915 Space Saver Plate Multiple Gel Caster 1 SE912 Glass Hinge Cassette 25 x 20 1 mm 1 SE9102 1 1 0 Plate Rack 1 SE914 Drain Filter 3 SE917 Gel Seal 1 SE6070 Related Products Isoelectric Focusing Unit 1 IEF100 Recirculating Chiller 1 RCB20 PS300B Power Supply 1 PS300B Acrylamide 1 kg GR141 1 Agarose 500 g GR140 500 Ammonium Persulfate 10 g GR152 10 Brilliant Blue G 258 GR134 25 Brilliant Blue R 258 GR135 25 Bromophenol Blue 10 g GR120 10 Dithiothreitol DTT 58 GR122 5 Glycerol 1L GR124 1 Glycine 1 kg GR125 1 N N Methylene bis Acrylamide 100 g GR142 100 Sodium Dodecyl Sulfate 500 g GR126 500 TEMED 258 GR151 25 Tris 1 kg GR132 1 Tris Glycine SDS Buffer 10X 1L GR149 1 Urea 1 kg GR143 1 e p23 e p24 Appendix A Theory and Recipes Laemmli System Gels The Laemmli system is the most common electrophoresis protocol for SDS denatured proteins The leading ion in this discontinuous buffer system is chloride and the trailing ion is glycine Accordingly the resolving gel and the optional stacking gel contain Tris Cl buffers of different concentration and pH and the electro phoresis buffer contains Tris glycine All buffers contain 0 1 SDS Polyacrylamide gel composition i
17. best top gel surface Slowly deliver the overlay solution on one side minimizing mixing and allow the overlay to flow across the top surface unaided 12 Allow the gels to polymerize for a minimum of one hour After polym erization pour off the overlay and rinse the gel surface several times with distilled water 13 Prepare the stacking gel monomer solution Pour the stacking gel on top of the resolving gel Allow a minimum of one hour for the stacking gel to polymerize p13 Note The Plate Rack SE914 is a convenient accessory for temporarily holding the gel cassettes when disassembling the multiple gel caster e p14 Disassembling the Multiple Gel Caster 1 Place the caster down on its back in a tray or sink 2 Remove the red clamps and screws securing the faceplate to the caster Slide off the faceplate Remove gel cassettes and filler sheets Rinse outside of the gel cassettes to remove excess polymerized acrylamide 5 Clean the multiple gel caster components with laboratory detergent Rinse and let air dry IPG Strip Equilibration Before IPG strips are placed on top of the second dimension gel the buffer in the strips needs to be replaced with an appropriate buffer for PAGE Equilibrate IPG strips in appropriate equilibration buffers Typically a two step equilibration first with DTT and a second with iodoacet amide gives better results than a single DTT equilibration step The following procedure is rec
18. degree 2 12 psi 43 x 43 x 20 cm 17 x 17 x 8 inch 22 3 lbs 10 1 kg 100 240 V 50 60 Hz 2A EN61010 1 2001 CE Fig 2 The SE900 The separation tank PAGE rack and safety lid Unpacking and Inventory Unwrap all packages carefully and compare the contents with the packing list making sure all the items have arrived If any part is missing contact your local sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Safety Lid SE9056 PAGE Rack SE9054 Separation Tank User Manual SE900 IM US Gel Seal SE6070 Wonder Wedge SE1514 lt gt Power Pack with 115 V Cord and 230 V EU amp UK Cords Tubing Kit Recirculation Bath SE908 Drain Filters SE917 The SE900 1 0 The system including the SE900 above multiple gel caster 6 glass cassettes Glass Cassette Multiple Gel Caster SE9102 1 1 0 SE915 e p3 Caution Never use water alcohol mixtures or commercial antifreeze as the coolant in the recirculating water bath This will cause irreparable damage to the separation tank Note The separation tank is chemically resistant to common electrophoretic buffers but not to organic solvents or strong acids and alkali Temperatures above 45 C may cause acr
19. dges This indicates current leakage at the spacers Make sure to clean the plates well at the spacer edges that the sides are free of dirt or grease and that the gel is fully polymerized before removing from the caster Gels have been run with hinge side toward the top of the tank The hinge also seals the lower edge of the gel from electrical leaks It is best to place cassettes hinge side down to get the straightest dye fronts Inadequate buffer at the top Make sure the buffer is filled Ya way through the top spacer to make the most even electrical field across the gel and prevent the gel from drying out Too much buffer at the top of the gel Make sure the buffer is filled V way through the top spacer to make the most even electrical field across the gel and prevent the current from bypassing over the gel at the top Check buffer recirculation is on and operating properly Use a refrigerated water bath to maintain even buffer temperature within the tank Decrease the current or voltage setting Dye front curves down frowns at the edges Poor polymerization at the spacers Make sure to clean the plates well at the spacer edges that the sides are free of dirt or grease and that the gel is fully polymerized before removing from the caster Power Supply detects current leak Cracked or broken alumina plate in the base of the separation tank Contact Hoefer or your distributor Poor draining of ta
20. dirt or grease and that the gel is fully polymerized before removing from the caster Gel is too soft too brittle or white Vertical protein streaks Adjust crosslinker concentration Crosslinker should be at 2 6 C for standard SDS gels where C g bis x 100 g monomer g bis Gel contains swirls Indicates convection currents during polymerization usually from polymerizing too fast If gel polymerized in lt 10 min too much catalyst Reduce concentration of ammonium persulfate and TEMED by 25 If gel polymerized in gt 50 min not enough catalyst Increase concentration of ammonium persulfate and TEMED by 50 Make sure gel solutions are near room temperature when casting Gels cast simultaneously are different sizes p20 Wait one minute before overlaying each gel so that the solution settles Use the same amount of overlay on each separation gel Add the overlay evenly across the gel surface Problem Solution Gradient gels uneven layering Add sucrose 15 final concentration or glycerol 25 final concentration to the high percent monomer solution Solutions pumped in too fast Add a small amount of bromophenol blue to the heavy solution to track the gradient formation Allow for solutions to underlay without excessive mixing Caution Excessive amounts of bromophenol blue inhibit polymerization Electrophoresis Run Problems Dye front curves up smiles at the e
21. e opl sningsmidler vil for rsage uboelig skade til enheden Driver ikke med st dpudetemperaturer over maksimummet specificerede tekniske specifications Overheding vil for rsage uboelig skade til enheden Belangrijke Informatie Dutch Indien deze uitrusting in een manier wordt gebruikt die niet door Hoefer Inc is gespecificeerd de bescherming die door de uitrusting is verzorgd kan worden geschaad Dit instrument is voor binnenlaboratoriumgebruik enkel ontworpen Enkel onderdelen en delen keurden goed of leverden door Hoefer Inc kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product gebruik Enkel een netvoeding die CE is markeerde of veiligheid die door een is gecertificeerd die nationaal is herkend testene laboratorium Het veiligheidsdeksel moet in plaats voor het verbinden van de netvoeding leidt tot een netvoeding zijn Doe alle netvoedingscontroles Uit en koppel los de machtleiding voor het verwijderen van het veiligheidsdeksel Circuleer enkel water of 50 50 water ethyleenglycol door de hitte exchanger zo ja uitrust Verbind de hitte exchanger naar een waterkraan of koelmiddelbron niet waar de waterdruk niet geregulariseerd is Stel Nooit antivriesmiddel of organische oplosmiddelen in deel van het instrument voor Organische oplosmiddelen zullen onherstelbare schade aan de eenheid veroorzaken Bedien niet met buffertemperaturen boven het maximum specificeerde technische specificaties
22. es Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este s mbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el equipo Denna symbol anger att elektriska och elektroniska utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information angaende avyttring av utrustningen e pv Fig 1 The Hoefer SE900 Function and Description The Hoefer SE900 vertical slab gel electrophoresis unit is intended for the second dimension of 2D electrophoresis It is designed for gel systems which use a single buffer within the gel tank such as those described by Laemmli Both buffers used in the anodic and cathodic chambers must be the same Up to 6 second dimension gel separations can be performed simultaneously The first dimension separation of 2 D protein electrophoresis sh
23. es de alimentaci n y desconecta los plomos del poder antes de quitar la tapa de la seguridad Circula s lo agua o 50 50 glicol de agua etileno por el intercambiador de calor si se es el caso equiparon No conecte el intercambiador de calor a un toque de la agua ni cualquier fuente del l quido refrigerante donde la presi n del agua est libre Nunca introduce anticongelante ni alg n solvente org nico en cualquier parte del instrumento Los solventes org nicos causar n da o irreparable a la unidad No opera con temperaturas de b fer encima del m ximo especific especificaciones t cnicas Recalentar causar da o irreparable a la unidad Viktig Information Swedish om denna utrustning anvands i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhandah ll vid utrustningen kan skadas Detta instrument formges f r inomhuslaboratorium anv ndning bara Bara medhj lpare och delar godk nde eller levererade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt anvander bara en kraft tillgang som ar CE markerade eller s kerhet intygade vid en nationellt erk nd testande laboratorium S kerheten locket m ste vara pa platsen fore koppla kraften tillgangen blyen till en kraft tillgang Vander sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen f re flytta s kerheten locket Cirkulerar bara vatten eller 50 50 vatten ethylene glycol genom varmen exchanger
24. he power leads before removing the safety lid Circulate only water or 50 50 water ethylene glycol through the heat exchanger if so equipped Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated Never introduce antifreeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperatures above the maximum specified technical specifications Overheating will cause irreparable damage to the unit Dulezit Informace Czech Pokud by toto za zen je pou ito zp sobem kter neni podle Hoefer Inc ochrana poskytovan na z klad za zen m e b t naru ena Tento n stroj je ur en pro vnit n pou it v laborato i pouze Pouze p slu enstv a sti schv len nebo poskytnut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku zdroj nap jen pou vaj jen e je opat en ozna en m CE osv d ena nebo bezpe nost vnitrost tn uznan mi zku ebn mi laborato Bezpe nosti lid mus b t zavedena p ed p ipojen m nap jec zdroj nap jen vede k Turn ve ker nap jen kontroly vypnuto a odpojit p ed odb rem energie vede bezpe nostn v ko Rozeslat pouze voda nebo 50 50 voda ethylenglykolu prost ednictv m v m n k tepla je li to vybavena Nemaj p ipojen v m n k tepla s vodn mi set epn nebo j
25. hecido testando laborat rio A tampa de seguranca deve estar em lugar antes de ligar o estoque de poder leva a um estoque de poder Desliga todos controlos de estoque de poder e desconecta os chumbos de poder antes de retirar a tampa de seguran a Circulam s gua ou 50 50 glicol de gua ethylene pelo exchanger de calor se for assim equiparam Nao ligue o exchanger de calor a uma torneira de gua nem qualquer fonte de refrigerante onde a press o de agua nao regulado Nunca introduz anticongelante nem qualquer org nico solvente em qualquer parte do instrumento Org nico solvente causar agress o irrepar vel unidade Nao opera com temperaturas de buffer acima do m ximo especificou especificac es t cnicas Superaquecer causar agress o irrepar vel unidade Informaci n Importante Spanish Si este equipo es utilizado en una manera no especificado por Hoefer Inc la protecci n proporcionado por el equipo puede ser da ada Este instrumento es dise ado para el uso interior del laboratorio s lo S lo accesorios y partes aprobaron o suministraron por Hoefer Inc puede ser utilizado para operar para mantener y para atender a este producto S lo utiliza una alimentaci n que es CE marc o la seguridad certificada por un nacionalmente reconocido probando el laboratorio La tapa de la seguridad debe estar en el lugar antes de conectar la alimentaci n lleva a una alimentaci n Apaga todos control
26. ight runs 18 hours set the power supply for 80 V constant The current and wattage should be set not to exceed 500 mA and 100 W 11 The run is complete when the tracking dye has reached the opposite side of the gels 12 Turn off the power supply the circulation pumps and the external recirculating water bath if being used epl7 Note DO NOT use the buffer circulation pumps to help the tank drain faster Fig 22 Drain port p18 After Electrophoresis 1 2 3 10 11 12 13 Unplug the leads to the power supply Remove the lid by moving the safety interlocks while lifting the lid Carefully lift out each gel The cassettes are slippery Do not use the PAGE rack as a gel carrying rack as the plates may slide out of the rubber gaskets when tipped or moved Place the glass cassette on a clean flat surface Use the Wonder Wedge to gently open the nonhinged side of the gel cassette Use the Wonder Wedge to gently cut the gel away from the side spacers Carefully remove the gel from the cassette and place in desired staining solutions Attach a length of tubing long enough to reach a sink to the drain connector Fig 22 Insert the connector into the drain port on the lower left side of the SE900 behind the black safety interlock Allow the tank to drain by gravity The tank will drain best by having the drain line at least 18 inches 50 cm below the tank Once drained re insert empty PAGE rack Fill the
27. ing flap seals closed at end of gel current cannot get to gel Fig 20 Gel orientation Fig 21 Slide safety lid into place Note The black high voltage lead should be on the same side as the IPG strip Red High Voltage Lead Red Safety Interlock Black High Voltage Lead Black Safety Interlock Proper Fill Level IPG Strip 1 1 1 1 1 1 4 Ll Rubber Hinge 7 After all gels are placed in the tank check the level of buffer in the separation tank The proper level is about halfway up the spacer on the top edge of the gel cassettte The buffer level also must be below the maximum fill line 8 Attach the safety lid to the separation tank in the proper orientation by moving and fitting the top of the safety interlocks through the slots on the lid and connecting the high voltage leads to the banana plugs The safety interlock will secure the lid in place Fig 21 9 Connect the high voltage leads to a power supply capable of delivering at least 300 V 500 mA 90 W such as the Hoefer PS300 B Adapters may be needed if using other power supplies 10 Set the power supply to the desired values and start the run Note For day runs set the power supply for 80 mA gel When running a full tank of 6 gels set the power supply for 480 mA Runs should be complete in about 6 hours The voltage and wattage should be set to their maximums 600 V and 100 W respectively so as not to limit the current For overn
28. lution needed Divide the total volume in half and prepare this volume of both the higher and lower percentage acrylamide solutions 4 Optional Adjust the higher percentage acrylamide solution to 15 w v sucrose or up to 25 v v glycerol to improve layering 5 Pour the higher percentage or heavy acrylamide solution into the SG500 chamber farthest from the outlet Open the stopcock just long enough to displace air between the chambers and then close 6 Pour the lower percentage or lighter acrylamide solution into the mixing chamber the chamber with the SG500 outlet 7 Place a stir bar into the mixing chamber Place the gradient maker onto a magnetic stirrer and begin stirring at a rate that mixes well but does not introduce bubbles into the solution 8 Turn on the peristaltic pump to a low setting and open the stopcock between the two SG500 chambers 9 The gel solution should slowly layer in the triangular region at the bottom of the multiple gel caster and fill the cassettes evenly from the bottom to the top 10 Once almost all solution has exited the gradient maker pause the pump temporarily and fill the gradient maker with 200 mls of the displacement solution Restart the pump and pump the solution through the tubing forcing the acrylamide solutions up into the gel cassettes until the desired height is reached Stop the pump 11 Overlay each gel with 1 ml of water saturated n butanol or diluted gel buffer to get the
29. nk Drain fitting has become clogged with debris Gently back flush water through the drain line Unusually long run times Too much buffer at the top of the gel Make sure the buffer is filled V way through the top spacer to make the most even electrical field across the gel and prevent the current from bypassing over the gel at the top Poor reagent quality Acrylamide solutions can build up acrylic acid over time Do not keep stock acrylamide solutions longer than 3 months and store acrylamide at 4 C Buffer incorrectly prepared Use the basic form of Tris Do not adjust the pH of the buffer after preparation Check power settings and adjust if necessary e p21 Problem Solution Instrument Problems No voltage or current at start of the run Instrument not properly attached to power supply Make sure high voltage leads are connected into correct terminals and is secure In some cases adapters may be required Broken electrode Check continuity of wire with a volt meter Lid not secured properly onto banana plugs Reposition and check lid is fit securely in place Break in HV lead line Check continuity of wire with a volt meter 2D Results Problems Vertical streaking of sample down from the top of the gel towards the bottom of the gel IPG Strip not properly placed on gel surface Avoid gouging separating gel while loading strips Perform iodoacetamide treatment Make s
30. ommended Equilibration Procedure 1 Thaw two aliquots of the equilibration solution 2 Add 10 mg ml DTT to one solution 3 Place the IPG strips in the rehydration equilibration tray or a glass tube Add 6 5 ml of solution to each slot containing an IPG strip Place on rocker for 10 15 minutes Following equilibration discard the first equilibration solution in an appropriate manner 6 Add 25 mg ml iodoacetamide IAA to the second aliquot of equilibration solution Add 6 5 ml of solution to each slot containing an IPG strip 8 Place on rocker for 10 15 minutes Following equilibration discard the second equilibration solution in an appropriate manner Following equilibration the IPG strips are placed on the top of the second dimension gel and sealed into place with the agarose overlay X 110 Sealing the IPG to the Second Dimension Gel 1 Remove residual liquid from the top of the second dimension gel by tipping the cassette and blotting one corner with a lint free tissue 2 Apply the equilibrated IPG strip to the gel surface Fig 17 A thin plastic strip is useful to gently seat the IPG directly against the top of the second dimension gel Do not press the strip into the gel or the results may be distorted Avoid trapping air bubbles between the strip and the second dimension gel 3 Seal the IPG strip in place by applying hot molten agarose over the Fig 17 Applying IPG strip to second IPG strip Fig 18 See
31. ory detergent Rinse thoroughly first with water and then with distilled water Glass plates should be washed well and dried completely before casting gels Plug one end of the power pack into the power entry on the lower right side of the SE900 Plug the other end into an appropriate grounded outlet The circulating pumps are turned on with a toggle switch on the lower right side of the SE900 Fig 13 Connect the cooling ports to an external recirculating water bath if active cooling is desired Note If desired labels printed on filter paper can be included in the cassette Sheets of 1 Whatman filter paper can be used to print gel id numbers cut into small pieces and positioned in the bottom corners of the cassette When gel solutions are added the numbers will be polymerized into the gel matrix allowing for easier identification of second dimension gels Caution Glass edges may be sharp so handle glass cassettes with care Fig 14 Multicaster assembly Using the Multiple Gel Caster Casting Homogenous Gels 1 Make sure the caster and the glass cassettes are clean and dry 2 Close each of the glass cassettes making sure the edges align flush 3 Disassemble the clean multiple gel caster and lay the back of the caster flat on the bench The two faceplate screws should be loosened but can remain in place The triangular gasket should be put in place Place the space saver in the back of the caster Place a separ
32. ould be performed on Immobilized pH Gradient Gels referred to as IPG strips in this manual The Hoefer IEF100 can be used to generate first dimension separations in which the proteins are separated by pI The focused strips are transferred to the SE900 second dimension slab gel for size separation The SE900 is offered as a self cast gel system The SE900 glass cassettes are 28 cm wide and 21 cm in length producing gels 25 cm wide x up to 20 cm tall and 1 mm in thickness The SE900 will also hold glass plates and gels from other vendors The SE900 is the separation tank as a stand alone unit The SE900 1 0 comprises the separation tank a multiple gel caster and six glass cassettes pl This declaration of conformity is only valid for the instrument when it is used in laboratory locations e used as delivered from Hoefer Inc except for alterations described in the user manual and e connected to other CE labeled instru ments or products recommended or approved by Hoefer Inc p2 Specifications Gel plate size w x h Gel size Maximum watt Maximum volt Maximum ampere Maximum temperature Environmental operating conditions Maximum recirculation water pressure Dimensions w x h x d Weight Input rating Product certifications 28 x 21 cm 25 x 20 cm 100 W 600 V 1000 mA 45 C Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category Il Pollution
33. produktu korzysta jedynie zasilacza e jest nosz ce oznakowanie CE lub bezpiecze stwa uwierzytelnione przez uznane na poziomie krajowym laboratorium badawcze Bezpiecze stwo lid musi by w miejsce przed pod czeniem zasilania prowadzi do zasilania Za wszystkie r d a zasilania urz dzenia steruj ce off i od czy moc prowadzi przed odbiorem bezpiecze stwa lid Kr tylko wody lub wody 50 50 ethylene glycol wymiennik ciep a poprzez je li tak wyposa one Nie nale y po czy wymiennik ciep a woda z kranu lub jakimkolwiek ch odziwo r d a je eli ci nienie wody jest nieuregulowanych Nigdy nie wprowadza rozpuszczalnika organicznego przeciw zamarzaniu lub jakichkolwiek na dowoln cz dokumentu Rozpuszczalniki organiczne spowoduje nieodwracalne szkody dla jednostki Nie dzia aj w buforze temperatury powy ej maksymalnego okre lone specyfikacje techniczne Przegrzania spowoduje nieodwracalne szkody dla jednostki Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protecc o fornecida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S6 acess rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto S usa um estoque de poder que CE marcou ou seguranca registrada por um nacionalmente recon
34. properly and the safety lid will not fit The electrodes terminate at banana plugs that connect to the safety lid Fig 7 Slots with rubber seals accept the gel cassettes and hold them verti cally in the separation tank If one of the six positions is empty the rubber seals eliminate electrical leaks without the need for blank cassettes or space fillers Caution Always install the safety lid before use Fig 8 Safety lid Fig 9 SE9102 1 1 0 glass cassette Safety Lid The safety lid holds the high voltage leads that connect to an external power supply not supplied The lid is held in place by the safety interlocks during electrophoresis Fig 8 The color coded high voltage leads have safe 4 mm plugs that inter face directly with Hoefer power supplies Adapters may be required to connect the SE900 to other power supplies Check the connection before using the SE900 Glass Cassettes The glass plates are 28 cm wide x 21 cm in length The cassettes have 1 mm thick glass spacers glued into place and are hinged for easy assembly Fig 9 Six glass cassettes are included with each SE900 1 0 Additional cassettes can be ordered separately as SE9102 1 1 0 It is important that the cassettes be positioned with the hinge side down in the separation tank during electrophoresis e p7 Fig 10 Multiple gel caster Fig 11 Multicaster filling line e p8 Multiple Gel Caster The
35. protezione fornito dall apparecchiatura potrebbe essere indebolita Questo strumento disegnato per l uso di laboratorio interno solo Solo gli accessori e le parti hanno approvato o hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare questo prodotto usa Solo un alimentatore che CE ha marcato o la sicurezza certificato da un nazionalmente riconosciuto testando il laboratorio Il coperchio di sicurezza deve essere nel luogo prima di collegare i piombi di alimentatore a un alimentatore Spegne tutto i controlli di alimentatore e disinserisce i piombi di potere prima di togliere il coperchio di sicurezza Circola solo l acqua o 50 50 glicole di acqua etilene attraverso lo scambiatore di calore se cosi equipaggiato Non collegare lo scambiatore di calore a un rubinetto di acqua o qualunque fonte di refrigerante dove la pressione di acqua sregolata Non introduce mai l antigelo o qualunque solvente organico in qualunque parte dello strumento solventi organici causeranno il danno irreparabile all unita Non opera con le temperature di tampone al di sopra del massimo ha specificato le descrizioni tecniche Il surriscaldamento causer il danno irreparabile all unita Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesifisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innend r
36. ps should be equilibrated just prior to second dimensin PAGE Do not equilibrate the IPG strips before storing at 20 C 9 1 Agarose in 1X Electrophoresis Buffer 100 ml In a 500 ml flask add Agarose lg 10X Electrophoresis Buffer Solution 8 10 ml Deionized H20 90 ml Bromophenol Blue 3 mg Gently swirl to suspend agarose Heat at low power in a microwave oven until agarose is fully dissolved Divide into 1 5 ml plastic screw top tubes Store in aliquots at 4 C 10 Gradient Gel Casting Displacement Solution 50 Glycerol in 0 375 mM TrisCl 8 8 0 1 SDS 200 ml Glycerol 1 5M TrisCI ph 8 8 Solution 2 Deionized H20 Bromophenol Blue Mix well 100 ml 50 ml 50 ml 3 mg 11 SDS Equilibration Buffer Solution This solution is used after IEF and before second dimension PAGE The IPG strips are immersed in excess solution to raise the pH of the strip buffer so that it is suitable for PAGE and to coat the proteins in SDS uniformly so that they migrate properly in the second dimension gel Prepares 200 ml 6 M urea 75 mM Tris HCl pH 8 8 29 3 glycerol 2 SDS 0 002 bromophenol blue Final Concentration Amount Urea FW 60 06 6M 72 1 1 5M Tris HCl pH 8 8 stock solution 75 mM 10 0 ml Glycerol 87 w w 29 3 v v 69 ml SDS FW 288 38 2 wiv 4 0 g Bromophenol blue 0 002 w v 4 mg Deionized H20 to 200 ml Aliquot into 30 ml aliquots and store frozen at 20 C or below 24 cm IPG
37. r Inc representative p19 Troubleshooting Problem Solution Casting Issues Gel caster leaks If the stack is too tall the front plate may not seat firmly against the gasket Remove filler plates or cassettes until the gasket seals Apply a light film of gel seal to the foam gasket each time the unit is used Check the foam gasket for cracks or nicks and replace if necessary Caster face plate not properly aligned Check that the faceplate is evenly positioned on caster Poor or Incomplete gel polymerization Use only recent stock of the highest quality reagents APS reagents or solutions are old and lose their activity when exposed to moisture Make up fresh APS daily If the dry ammonium persulfate does not crackle when water is added to it replace the product with fresh stock Store reagent tightly closed and in a dessicator to prevent absorption of moisture Remove oxygen from the gel environment Degas the monomer solution 5 minutes before pouring and then overlay the gel surface with water saturated n butanol Allow gel solutions to come to room temperature before casting a minimum of 20 C especially for low T gels Increase both APS and TEMED by 30 50 Solutions with extreme pH values especially acidic may not polymerize Check pH of gel buffers Poor polymerization at the spacers Make sure to clean the plates well at the spacer edges that the sides are free of
38. rch die Ausr stung zur Verf gung gestellte Schutz verschlechtert werden Dieses Instrument wird f r den Innenlaborgebrauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durch Hoefer Inc kann f r das Funktionieren das Aufrechterhalten und die Wartung dieses Produktes verwendet werden Verwenden Sie nur eine Energieversorgung die CE gekennzeichnet oder durch ein national anerkanntes Probelaboratorium bescheinigte Sicherheit ist Der Sicherheitsdeckel muss im Platz vor dem Anschlie en der Energieversorgung sein f hrt zu einer Energieversorgung Alle Energieversorgungssteuerungen abdrehen und die Macht trennen f hrt vor dem Entfernen des Sicherheitsdeckels Nur Wasser oder 50 50 Glykol des Wassers Athylens durch den W rmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder K hlmittel Quelle nicht wo der Wasserdruck ungeregelt wird F hren Sie nie Frostschutzmittel oder jedes organische L sungsmittel in jeden Teil des Instrumentes ein Organische L sungsmittel werden nicht wiedergutzumachenden Schaden der Einheit verursachen Mit Puffertemperaturen ber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die berhitzung wird nicht wiedergutzumachenden Schaden der Einheit verursachen Informazioni Importanti Italian Se quest apparecchiatura usata in un modo specificato da Hoefer Inc la
39. s indicated by two different percentages T total acrylamide g acryl bis x 100 100 96 C crosslinker g bis x 100 g acryl bis The total percent of acrylamide T in the resolving gel which can range from 5 to 20 determines the pore size Commonly the amount of crosslinker used C is 2 6 Caution Acrylamide is a neurotoxin Extreme care should be used when handling and preparing acrylamide solutions Solutions 1 Acrylamide Stock Solution 30 8 T 2 6 C Bis 1000 ml Acrylamide FW 71 08 30 wiv 300 g Bis N N Methylenebisacrylamide FW 154 2 0 8 wiv 8g Deionized H20 to 1000 ml Store at 4 C away from light 2 1 5 M TrisCl pH 8 8 4X Resolving gel buffer 2 liter Tris FW 121 1 1 5 M 363 3 g Disolve into 1 5 liters deionized H20 4 N HCI AN hydrochloric acid to pH 8 8 Deionized H20 to 2000 ml Store at 4 C 3 1096 w v SDS Solution 100 ml Sodium dodecylsulfate SDS FW 288 4 0 35 M 10g Deionized H20 to 100 ml Store at room temperature 4 1096 w v APS Initiator 1 ml Ammonium persulfate APS FW 228 2 0 44 mM 5g Deionized H20 to 5 ml Prepare just prior to use Fresh APS crackles when water is added If yours does not replace it with fresh stock 5 10 v v TEMED Catalyst 5 ml N N N N Tetramethylethylene diamine TEMED FW116 2 0 65 M 0 5 ml Deionized H20 to 5 ml Prepare just prior to use Store in a dark glass bot
40. s laboratoriumbruk bare Bare tilbehor og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt for drive vedlikeholde og betjene dette produktet bruker Bare en kraftforsyning som er CE merket eller sikkerhet som ha blitt sertifisert av et som nasjonalt ha blitt anerkjent prover laboratorium Sikkerheten lokket ma vaere pa plass for forbinding kraftforsyningene blyene til en kraftforsyning Vender all kraftforsyningsstyring av og frakopler kreftene blyene for fjerning sikkerheten lokket Sirkulerer bare vann eller 50 50 vann ethylene glykol gjennom oppvarmingen veksleren i s fall utstyrer Ikke forbind oppvarmingen veksleren til en vanntapp eller noe kj lemiddelkilde hvor vannet e piii trykket er unregulated Introduserer Aldri antifreeze eller noe organisk losemiddel inn i noe del av instrumentet Organiske losemiddler vil for rsake irreparabel skade p enheten Driver med buffertemperaturer over maksimum ikke spesifiserte teknisk spesifikasjoner A overoppheting vil for rsake irreparabel skade p enheten Wazne Informacje Polish Je eli ten sprz t jest wykorzystywany w spos b nie okre lone przez Hoefer Inc do ochrony przewidzianej przez urz dzenie mo e zosta obni ony Instrument ten jest przeznaczony do u ytku w laboratoriach kryty tylko Tylko akcesori w i cz ci zatwierdzone lub dostarczone przez Hoefer Inc mog by wykorzystane do eksploatacji utrzymania i obs ugi tego
41. s require 5 10 ml per strip per equilibration step Shorter strips can use proportionately less volume per equilibrations step e p27 p28 Gel Recipes The Laemmli gel recipes are for 400 ml of a single concentration solu tion enough for six 1 0 mm 25 x 20 cm gels Tabulated are volumes for relatively large pore gels 10 T range as well as smaller pore gels 12 5 20 T range 5 and 7 5 gels are difficult to handle in large format gels but can be blended into gradient gels for better reso lution and easier handling The recipes given here are for guidance when casting gradient gels Use L total required volume each solution when casting gradient gels Using the solutions given in Appendix A all gels are crosslinked with 2 6 C Laemmli Gel per 400 ml gel solution Resolving Gel Solution 400 ml 10 0 12 5 15 0 17 5 20 Acrylamide Stock Soln 1 133 3 166 7 200 0 233 3 266 7 1 5 M TrisCl pH 8 8 Soln 2 100 0 100 0 100 0 100 0 100 0 10 SDS Soln 3 4 0 4 0 4 0 4 0 4 0 Deionized H20 158 0 1248 91 5 583 25 0 10 APS Soln 4 4 0 4 0 4 0 4 0 4 0 10 TEMED Soln 5 0 68 0 55 0 45 0 40 0 34 Final Volume 400 0 400 0 400 0 400 0 400 0 Laemmli Gel per 200 ml each gel solution Gradient Gels Solution 200 ml LIGHT 5 0 7 5 10 0 12 5 15 0 17 5 Acrylamide Stock Soln 1 33 3 50 0 66 7 83 3 100 0 116 7 1 5 M TrisCl 50 0 50 0 50 0 50 0 50 0 50 0 pH 8 8 Soln 2 10 SDS Soln 3 2 0 2 0 2 0
42. tle at room temperature away from light TEMED is flammable and should be dispensed in a fume hood e p25 p26 6 0 375 M Tris Cl 0 1 SDS pH 8 8 Resolving gel overlay 100 ml 1 5 M Tris Cl pH 8 8 Solution 2 0 375 M 25 ml 10 SDS Solution 4 3 5 mM 1 ml Deionized H20 to 100 ml Store at room temperature Water saturated n butanol Shake n butanol and deionized H20 in a separatory funnel Remove the aqueous lower phase Repeat this procedure several times Use the upper phase Store at room temperature 7 10X Electrophoresis Buffer 0 25 M Tris 1 92 M Glycine 1 0 SDS 10X Electrophoresis buffer 5 0 liters Add powders slowly to 4 liters deionized water while stirring Tris FW 121 1 0 25 M 151 2g Glycine FW 75 07 1 92 M 720 6g SDS FW 288 4 35 mM 50 0 g Deionized H20 to 5 0 liters The pH of this buffer is approximately 8 3 Do not adjust pH Store room temperature for up to 2 months 8 1X Electrophoresis Buffer 0 025 M Tris 0 192 M Glycine 0 1 SDS 1X Electrophoresis buffer 12 0 liters 10X Electrophoresis Buffer Solution 7 1200 ml Deionized H20 10 8 This can be prepared in the separation tank Add 10X stock water then the PAGE rack Gently raise and lower the PAGE rack to aid in mixing then allow the recirculation system to mix the buffer well before use Caution SDS may cause the solution to boil over so exercise caution when heating and prevent boiling over Note IPG stri
43. ure IPG strip uniformly contacts the gel surface along the entire length of the strip Overloading of protein Horizontal streaking of proteins Poor sample preparation Inadequate focusing Poor contact between the IPG strip and the second dimension gel Spots are skewed or distorted Gels run too fast uneven migration Overlay the resolving gel with water saturated n butanol before polymeriza tion begins to avoid forming an uneven gel surface Uneven gel polymerization or gradient formation See casting problems for more support IPG strip not properly placed on gel surface Avoid gouging separating gel while loading strips Sample entry into the second dimension gel is run at too high a power setting Run gels at recommended run conditions Bubbles present in second dimension gel will distort the spot migration No proteins visible on second dimension gel after staining Quantity loaded too little for detection method Increase protein load or try a more sensitive staining method Too little sample applied to first dimension gel Check protein concentration of sample Equilibration steps too short or too long Perform each equilibration step for 10 15 minutes Blank regions in the second dimension gel Tris salts and SDS can cause alterations in the focusing position of proteins in the first dimension Reduce or eliminate these compounds from the first dimension Bu
44. ylic to warp Note There is no need for a magnetic stir bar The pumps circulate the buffer evenly Fig 3 Separation tank Note A light layer of gel seal has been applied inside the guide channels in the separation tank to allow the PAGE rack to slide into the tank easily Do not wash off Reapply as needed see Instructions page 9 Gel Seal e p4 Separation Tank The Separation tank is transparent to allow visualization of the track ing dyes during electrophoresis The chamber base contains a ceramic cooling surface which helps to efficiently cool the buffer within the tank Care should be taken not to drop ANYTHING directly on the ceramic plate A pump system forces buffer across the cooling surface and circulates the buffer through the center region of the tank maintaining a constant temperature in the gel cassettes Cooling ports can be attached to a temperature regulated recirculating water bath for active cooling of the separation tank The recirculating water bath should have a maximum output pressure of 12 psi Use ONLY water or up to a 50 mixture of ethylene glycol in water in the recirculating bath Never connect to an unregulated source of water such as a tap water PAGE Rack Channel Fig 4 Key aligns with a mating feature in the PAGE rack to ensure proper orientation Safety Interlocks Safety interlocks are mounted on the sides of the tank One is red and the other is black to help with orienting
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